Prostaglandin E receptors, their DNA and production

ABSTRACT

Disclosed are (1) a protein capable of receiving PGE, (2) a recombinant DNA coding for said protein, (3) a vector having said DNA, (4) a transformant carrying said vector, and (5) a method for producing said protein wherein said transformant is cultured in a culture medium, the protein being useful not only in cloning other PGE receptor genes, clarifying the structure of PGE receptors and elucidating the function of PGE, but also in searching for PGE antagonists and agonists and so on.

This application is a continuation of application Ser. No. 08/024,179,filed Feb. 23, 1993, now abandoned.

FIELD OF THE INVENTION

This invention relates to a PGE (especially PGE₂) receptor which bindsto prostaglandin (PG) E, particularly prostaglandin E₂ (PGE₂), which isknown to be involved widely in digestive tract constriction andrelaxation, gastric acid and intestinal juice secretion, smooth musclerelaxation, neurotransmitter release and other phenomena in vivo, on thecell membrane, and transmits information on PGE₂ etc. to cells, and agene which codes therefor.

BACKGROUND OF THE INVENTION

The importance of PGE, particularly PGE₂, in vivo is widely recognized.Analyses of the physiological and pharmacological action of PGE₂ andaction sites have suggested that there exist at least three types of PGEreceptors, EP₁, EP₂ and EP₃ and they are thought to be different intheir signal transduction. These subtypes are presumed coupled tostimulation of phospholipase C, and stimulation and inhibition ofadenylate cyclase, respectively. (R. A. Coleman, I. Kennedy, P. P. A.Humphrey, K. Bunce and P. Lumley, Comprehensive Medicinal Chemistry, ed.C. Hansch, P. G. Sammes and J. B. Taylor, Vol. 3, pp. 643-714, PergamonPress, 1990 and Annu. Rev. Pharm. Tox. 10, 213-239 (1989)). Among PGEreceptor subtypes, the EP₂ receptor has been suggested to be involved inrelaxation in trachea (Br. J. Pharmacol. 87, 45 (1986)) and ileumcircular muscle (Br. J. Pharmacol. 105, 271-278 (1992)), vasodilatationin various blood vessels, and stimulation of sodium and waterreabsorption in kidney tubulus (J. Clin. Invest. 47, 1154-1161 (1968)and J. Biol. Chem. 263, 6155-6160 (1988)). One of the most importantfunctions of PGE₂ through EP₂ receptor has been proposed to be negativeregulation of immune system (Am. Rev. Respir. Dis. 135, 72-77 (1987))and inflammation, and the EP₃ receptor has been suggested to be involvedin such PGE₂ actions as inhibition of gastric acid secretion (Chen etal., 1988. Gastroenterology 94, 1121-1129), modulation ofneurotransmitter release (Hedqvist et al., 1972. Neuropharmacology 11,177-187; Ohia and Jumblatt, 1990. J. Pharmacol. Exp. Ther. 255, 11-16),inhibition of lipolysis in adipose tissue (Richelsen et al., 1984. J.Lipid Res. 26, 127-134), and inhibition of sodium and water reabsorptionin kidney tubulus (Garcia-Perez et al., 1984. J. Clin. Invest. 74,68-74). However, no PGE₂ receptor genes have been successfully cloned;their distribution, structure and function remain to be investigated.There has been urgent demand for their elucidation for the purpose ofclarifying PGE-associated, particularly PGE₂ -associated diseases, andfor developing effective pharmaceuticals for such diseases.

OBJECT OF THE INVENTION

As stated above, the nature of the PGE receptor remains largely unknown.However, if a gene which codes for at least one type thereof issuccessfully cloned to yield a transformant which constantly expressesthe PGE receptor, cloning of the genes of other types of PGE receptorand structural determination of the PGE receptor will be feasible, butalso the role of PGE, particularly PGE₂ in vivo will be clarified. It isalso expected that receptor-specific antibodies will be successfullyobtained by using a transformant which expresses the receptor as animmunogen, which will contribute to elucidation of the histologicdistribution of receptor-expressing cells.

Generally, prostaglandin (PG) receptors are thought to be highlyhomologous to each other with respect to the amino acid sequence of theligand-binding site. It is therefore conjectured that part of the DNAsequence of the human thromboxane (TX) A₂ receptor gene [M. Hirata etal., Nature, 349, 617 (1991)] is very similar to the DNA sequence of thegene of the receptor of PGE such as PGE₂. Based on this idea, thepresent inventors succeeded in cloning from mouse cells a gene whichcodes for the EP₃ receptor, a subtype of mouse PGE₂ receptor, by usingpart of the human TXA₂ receptor gene as a probe, and further in cloningfrom mouse cells a gene which codes for the EP₂ receptor, a subtype ofmouse PGE₂ receptor, by using part of the mouse EP₃ receptor gene as aprobe. The present inventors constructed their recombinant DNAcontaining each of said genes, and then found that the transformantsresulting from transformation with each of said DNAs, whether entirelyor partially, is capable of binding to PGE₂.

The present inventors made further investigations based on thesefindings, and developed the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide (1) a protein capableof receiving PGE, (2) a recombinant DNA containing a gene which codesfor the protein of (1) above, (3) a vector containing the recombinantDNA of(2) above, (4) a transformant carrying the vector of (3) above,and (5) a method of producing the protein of (1) above wherein thetransformant of (4) above is cultured.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the base sequence of the clone MP660 containing the genewhich codes for the PGE₂ receptor, and the amino acid sequence deducedtherefrom. The Figure includes DNA sequence SEQ ID NO:1 and amino acidsequence SEQ ID NO:2.

FIGS. 2(a) and 2(b) show the inhibitory activities of various ligands onthe binding of [³ H]-PGE₂ to the PGE₂ receptor (EP₃) expressed onMP660-transfected COS-1 cell membranes.

FIG. 3 shows the inhibitory activities of various ligands on the cAMPsynthesis in CHO cells expressing the PGE₂ receptor.

FIG. 4 shows the base sequence of the clone MP653 containing the genewhich codes for the PGE₂ receptor, and the amino acid sequence deducedtherefrom. The figure includes DNA sequence SEQ ID NO:3 and amino acidsequence SEQ ID NO:4. The boxed portion of FIG. 4 designated"peptide-α"is present only in MP660, and is included in FIG. 4 forcomparison with MP653.

FIG. 5 shows comparison of cDNA structures of two EP₃ receptor isoforms.(Schematic representation of mouse EP₃ receptor cDNA clones, MP660 andMP653. Boxes represent coding sequences; open box is a correspondingcoding sequence between the two cDNA, grey one is the sequence codingpeptide-α, hatched one is the sequence coding peptide-β. The putativetransmembrane domains are indicated by striped boxes.)

FIG. 6 shows the base sequence of the clone MP412 containing the genewhich codes for the PGE₂ receptor, and the amino acid sequence deducedtherefrom. The figure includes DNA sequence SQ ID NO:5 and amino acidsequence SEQ ID NO:6.

FIG. 7 and 8 show the inhibitory activities of various ligands on thebinding of [³ H]-PGE₂ to the PGE₂ receptor (EP₂) expressed onMP412-transfected COS-1 cell membrances.

FIG. 9 shows the effect of PGE₂ on cAMP level in MP412-transfected oruntransfected COS-1 cells.

DESCRIPTION OF THE PREFERRED EMBODIMENT

PGEs for the present invention include PGE₁ and PGE₂, with preferencegiven to PGE₂. The capability of receiving PGE₂ means that the receptoris capable of specifically binding to PGE₂ or a similar substance in thetransmembrane domain, and that the structural change due to such ligandbinding induces activation of related GTP-binding protein in theintracellular domain.

The protein of the present invention, which is capable of receiving PGE(hereinafter also referred to as PGE receptor), is preferably a proteinwhich is capable of receiving PGE₂ (hereinafter also referred to as PGE₂receptor), and may be of several types, including human, chicken andmouse, and may be a glycoprotein resulting from sugar chain binding tothe sugar-binding site or a complex protein such as phosphoproteinresulting from phosphorylation at the phosphorylation site.

PGE₂ receptors of the mouse type include a polypeptide having the aminoacid sequence comprising a series of the amino acid sequence of FIG. 1(SEQ ID NO:2), a polypeptide having the amino acid sequence comprising aseries of the amino acid sequence of FIG. 4 (SEQ ID NO:4) and apolypeptide having the amino acid sequence comprising a series of theamino acid sequence of FIG. 5. Any PGE₂ receptor is acceptable, as longas it is capable of receiving PGE₂ and activating GTP-binding protein.The receptor-G protein coupling may be examined in several ways. Oneexample is to examine modulation of the finding affinity of the receptorby guanine nucleotides (Ann. Rev. Biochem. 56, 615-649 (1987)).Specifically, it may be a mutein resulting from deletion of at least oneconstituent amino acid from said amino acid sequence, replacement of atleast one constituent amino acid by another amino acid, or addition ofat least one amino acid, and may be a functional fragment.

The PGE receptor subtype may be of EP₁, EP₂ or EP₃ (α,β), withpreference given to the EP₂ receptor and the EP₃ receptor.

The gene which codes for the PGE receptor may be any one, as long as itcodes for the PGE receptor. For example, the gene which codes for theEP₃ α receptor, a mouse PGE₂ receptor subtype, the gene which codes forthe EP₃ β receptor, a mouse PGE₂ receptor subtype and the gene whichcodes for the EP₂ receptor, a mouse PGE₂ receptor subtype areexemplified by a gene having the base sequence comprising a series ofthe 1-1095 bases shown in FIG. 1 SEQ ID NO:1), a gene having the basesequence comprising a series of the 1-1083 bases shown in FIG. 4 (SEQ IDNO:3) and a gene having the base sequence comprising a series of the1-1539 bases shown in FIG. 5, respectively.

The vector according to the present invention, which harbors arecombinant DNA containing a gene which codes for PGE receptor, can, forexample, be produced by:

(1) separating the RNA which codes for the PGE receptor,

(2) synthesizing a single-stranded complementary DNA (cDNA) and then adouble-stranded DNA from said RNA,

(3) inserting said double-stranded DNA to a plasmid,

(4) transforming a host with the thus-obtained recombinant plasmid,

(5) cultivating the thus-obtained transformant and then isolating theplasmid containing the desired DNA therefrom by an appropriate method(e.g., colony hybridization using a DNA probe),

(6) cleaving out the desired cloned DNA from said plasmid, and

(7) ligating said cloned DNA to the downstream of the promoter in thevehicle.

Said cDNA can also be produced by chemical synthesis.

The RNA which codes for the PGE receptor can be obtained from variousPGE-receptor-expressing cells known to those of ordinary skill in theart, such as mouse mastocytoma line P-815 cells and IL-3 dependent cellline BNu-c13 cells.

Methods of preparing RNA from PGE-receptor-expressing cells include theguanidine thiocyanate method [J. M. Chirgwin et al., Biochemistry, 18,5294 (1979)]. Other suitable methods known to those skilled within thisart are also within this invention.

Using the thus-obtained RNA as a template in combination with reversetranscriptase, a cDNA is synthesized in accordance with, for example,the method of H. Okayarea et al. [Molecular and Cellular Biology, 2, 161(1982) and 3, 280 (1983)], and the resulting cDNA is inserted to aplasmid. Other methods for recombinantly synthesizing cDNA, known tothose skilled in the art are also within the claimed invention.

Examples of the plasmid for cDNA insertion include plasmids derived fromEscherichia coli such as pBR322 [Gene, 2, 95 (1977)], pBR325 [Gene, 4,121 (1978], pUC12 [Gene, 19, 259 (1982)], pUC13 [Gene, 19, 259 (1982)],pUC118 and pUC119 [Methods in Enzymology, 153, 3-11 (1987)] and thosederived from Bacillus subtitis such as pUB110 [Biochemical andBiophysical Research Communications, 112, 678 (1983)], but any other canbe used for this purpose, as long as it is replicable in the host.

Examples of the method of insertion to the plasmid include thatdescribed by T. Manjarls et al. in Molecular Cloning, Cold Spring HarborLaboratory, page 239 (1982).

The plasmid incorporating said eDNA may be a plasmid obtained by using acDNA library with Escherichia coli x1776 host prepared by inserting acDNA synthesized from human normal diploid cell mRNA to the pCD vector[see Okayarea et al., Molecular Cell Biology, 3, 280 (1983)], which cDNAlibrary is available from Dr. Okayama at the Research Institute forMicrobial Diseases, Osaka University.

The plasmid thus obtained is introduced to an appropriate host such as abacterium of the genus Escherichia or Bacillus.

Example bacteria of the genus Escherichia include Escherichia coliK12DH1 [Proceedings of the National Academy of Science, USA, 60, 160(1968)], M103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal ofMolecular Biology, 120, 517 (1978)], HB101 [Journal of MolecularBiology, 41, 459 (1969)] and C600 [Genetics, 39, 440 (1954)].

Example bacteria of the genus Bacillus include Bacillus subtilis MI114[Gene, 24, 255 (1983)] and 207-21 [Journal of Biochemistry, 95, 87(1984)].

Methods of transformation include the calcium chloride method andcalcium chloride/rubidium chloride method described by T. Maniatis inMolecular Cloning, Cold Spring Harbor Laboratory, page 249 (1982).

From the transformants thus obtained, the desired clone is selectedusing a known method, such as colony hybridization [Gene, 10, 63 (1980)]or DNA base sequencing [Proceedings of the National Academy of Science,USA, 74, 560 (1977); Nucleic Acids Research, 9,309 (1981)].

A microorganism carrying a vector having a cloned DNA containing a basesequence which codes for the PGE receptor is thus obtained.

Next, the plasmid is isolated from the microorganism.

Methods of such isolation include but are not limited to the alkalimethod [H. C. Birmboim et al., Nucleic Acids Research, 1, 1513 (1979)].

The above plasmid having a cloned recombinant DNA containing a genewhich codes for the PGE receptor can be used as such or after beingcleaved out with restriction enzyme as necessary.

The cloned gene is ligated downstream of a promoter, in a vehicle(vector) suitable for its expression, to yield an expression vector.

Example vectors include the above-mentioned plasmids derived fromEscherichia coli (e.g., pBR322, pBR325, pUC12, pUC13, pUC118, pUC119),plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194),yeast-derived plasmids (e.g., pSH19, pSH15), bacteriophages such as λphage, animal viruses such as retrovirus and vaccinia virus and plasmidsfor animal expression (e.g., pcDNAI, pdKCR-dhfr).

The gene may have ATG (base sequence which codes for an appropriatesignal peptide as desired) as a translational initiation codon at its5'-terminal and TAA, TGA or TAG (preferably TGA) as a translationaltermination codon at its 3'-terminal. To express the gene, a promoter isligated to the upstream thereof. Any promoter can be used for thepresent invention, as long as it is appropriate for the host used toexpress the gene.

Examples of preferred promoters include the T7 promoter, trp promoter,lac promoter, rec A promoter, λPL promoter or lpp promoter, when thetransformation host is a bacterium of the genus Escherichia; the SPO1promoter, SPO2 promoter or pen P promoter when the host is a bacteriumof the genus Bacillus; and the PHO5 promoter, PGK promoter, GAP promoteror ADH promoter when the host is a yeast. Preference is given to thecase in which a bacterium of the genus Escherichia is used as host incombination with the trp promoter or T7 promoter.

When the host is an animal cell, preferable promoters include theSV40-derived promoter and retrovirus promoter, with preference given tothe SV40-derived promoter.

The thus-constructed vector, harboring a DNA, is used to produce atransformant.

Examples of the host include bacteria of the genus Escherichia, bacteriaof the genus Bacillus, yeasts and animal cells. Examples of the bacteriaof the genus Escherichia and of the genus Bacillus include the same asspecified above.

Examples of the yeasts include Saccharomyces cerevisiae AH22R, NA₈₇₋₁₁ Aand DKD-5D.

Example animal cells include simian cells COS-7, Vero, Chinese hamsterovarian cells CHO, mouse L cells and human FL cells.

The bacteria of the genus Escherichia can be transformed in accordancewith the method described in the Proceedings of the National Academy ofScience, USA, 69, 2110 (1972), Gene, 17, 107 (1982) and otherpublications known to those skilled in the art.

Bacteria of the genus Bacillus can be transformed in accordance with themethod described in Molecular and General Genetics, 168, 111 (1979) andother publications, for instance.

Yeasts can be transformed in accordance with the method described in theProceedings of the National Academy of Science, USA, 75, 1929 (1978),for instance.

Animal cells can be transformed in accordance with the method describedin Virology, 52, 456 (1973), for instance.

A transformant resulting from transformation with a vector harboring thecDNA of PGE receptor is thus obtained.

For cultivating a transformant whose host is a bacterium of the genusEscherichia or Bacillus, it is appropriate to use a liquid mediumsupplemented with carbon sources, nitrogen sources, minerals and othersubstances necessary for the growth of the transformant. Example carbonsources include glucose, dextrin, soluble starch and sucrose. Examplenitrogen sources include organic or inorganic substances such asammonium salts, nitrates, corn steep liquor, peptone, casein, meatextract, soybean cake and potato extract. Example minerals includecalcium chloride, sodium dihydrogen phosphate and magnesium chloride.Yeasts, vitamins, growth promoters and other additives may be added.

The pH of the medium is preferably about 6 to 8.

Examples of media preferably used to cultivate Escherichia bacteriainclude the M9 medium containing glucose and casamino acid [Miller,Journal of Experiments in Molecular Genetics, 431-433, Cold SpringHarbor Laboratory, New York (1972)]. To increase promoter efficiency asnecessary, a chemical agent such as 3β-indolyl acrylic acid may beadded.

When the host is a bacterium of the genus Escherichia, cultivation isnormally carried out at about 15° to 43° C. for about 3 to 24 hours,with aeration and/or stirring as necessary.

When the host is a bacterium of the genus Bacillus, cultivation isnormally carried out at about 30° to 40° C. for about 6 to 24 hours,with aeration and/or stirring as necessary.

Examples of media for cultivating a transformant whose host is a yeastinclude Burkholder's minimal medium [Bostian, K. L. et al., Proceedingsof the National Academy of Science, USA, 77, 4505 (1980)]. It ispreferable to adjust the medium to a pH of about 5 to 8. Cultivation isnormally carried out at about 20° to 35° C. for 24 to 72 hours, withaeration and/or stirring as necessary.

Examples of media for cultivating a transformant whose host is an animalcell include MEM medium [Science, 122, 501 (1952)], DMEM medium[Virology, 8, 396 (1959)], RPMI1640 medium [Journal of the AmericanMedical Association, 199, 519 (1967)] and 199 medium [Proceedings of theSociety for the Biological Medicine, 73, 1 (1950)]. These media may besupplemented with about 5 to 20% fetal bovine serum. The pH ispreferably about 6 to 8. Cultivation is normally carried out at about30° to 40° C. for 15 to 60 hours, with aeration and/or stirring asnecessary.

Separation and purification of PGE receptor of the present inventionfrom the culture described above can, for example, be achieved asfollows:

In extracting the PGE receptor of the present invention from culturedbacterial, yeast or animal cells, the cells are collected by a knownmethod after cultivation and suspended in a buffer containing a proteindenaturant, such as guanidine hydrochloride, to elute the desired PGEreceptor extracellularly. In another method, the cells are disrupted byultrasonication, lysozyme treatment and/or freeze-thawing, after whichthey are centrifuged to separate the PGE receptor of the invention. Themethod using a combination of lysozyme and ultrasonication is preferred.

For purifying the PGE receptor of the present invention from thesupernatant, known methods of separation and purification can be used incombination as appropriate. Such known methods of separation andpurification include those based on solubility differences such assalting-out and solvent precipitation, those based mainly on molecularweight differences such as dialysis, ultrafiltration, gel filtration andSDS-polyacrylamide gel electrophoresis, those based on chargedifferences such as ion exchange chromatography, those based on specificaffinity such as affinity chromatography, those based on hydrophobicitydifferences such as reverse-phase high performance liquidchromatography, and those based on isoelectric point differences such asisoelectric focusing.

The thus-obtained PGE receptor of the present invention may be preparedas a dry powder by dialysis and lyophilization. It is appropriate to addserum albumin etc. as a carrier in storing the PGE receptor, since itsadsorption to the container is prevented.

The PGE receptor of the present invention, substantially pure, is thusobtained. The substantially pure protein of the present invention has aprotein content of not less than 95% (w/w), preferably not less than 98%(w/w).

The PGE receptor thus obtained itself, or a transformant expressing itor a moiety thereof can be used to screen substances exhibitingantagonistic or agonistic activity thereon by, for example, aligand-binding test. The PGE receptor, as such, can also be used as aPGE-masking protein. The transformant obtained according to the presentinvention, which expressed the PGE receptor, and parts thereof can beefficiently used to obtain antibodies against said receptor.

Abbreviations for bases, amino acids, solvents and others used in thepresent specification and drawings attached thereto are based onabbreviations specified by the IUPAC-IUB Commission on BiochemicalNomenclature or abbreviations in common use in relevant fields. Someexamples are given below. When an optical isomer may be present in aminoacid, it is of the L-configuration, unless otherwise stated. Theseabbreviations may represent residues of corresponding compounds capableof forming a peptide bond.

DNA: Deoxyribonucleic acid

cDNA: Complementary deoxyribonucleic acid

A: Adenine

T: Thymine

G: Guanine

C: Cytosine

RNA: Ribonucleic acid

mRNA: Messenger ribonucleic acid

dATP: Deoxyadenosine triphosphate

dTTP: Deoxythymidine triphosphate

dGTP: Deoxyguanosine triphosphate

dCTP: Deoxycytidine triphosphate

ATP: Adenosine triphosphate

EDTA: Ethylenediaminetetraacetic acid

SDS: Sodium dodecyl sulfate

Gly or G: Glycine

Ala or A: Alaninc

Valor V: Valine

Leu or L: Leucine

Ile or I: Isoleucine

Ser or S: Serine

Thr or T: Threonine

Cys or C: Cysteine

Met or M: Methionine

Glu or E: Glutamic acid

Gln or Q: Glutmine

Asp or D: Aspartic acid

Lys or K: Lysine

Arg or R: Arginine

His or H: Histidine

Phe or F: Phenylalanine

Tyr or Y: Tyrosine

Trp or W: Tryptophan

Pro or P: Proline

Asn or N: Asparagine

The present invention is hereinafter described in more detail by meansof the following examples, which are not to be construed as limitativeto the present invention.

The following clone cell lines which were obtained in the Examplesmentioned below were deposited at the Institute-for Fermentation, Osaka,Japan (IFO), and at the Fermentation Research Institute (NationalInstitute of Bioscience and Human-Technology), Agency of IndustrialScience and Technology, Ministry of International Trade and Industry,Japan (FRI) under the Budapest Treaty.

Their accession numbers on the deposit dates are shown in Table 1 below(The deposit dates are indicated in parenthesis)

                  TABLE 1                                                         ______________________________________                                        Clone Cell                                                                    line        IFO          FRI                                                  ______________________________________                                        MP660/KCR   IFO 50366    FERM BP-3803                                         (Example 1) (March 11, 1992)                                                                           (March 18, 1992)                                     MP653/KCR   IFO 50397    FERM BP-4183                                         (Example 2) (January 28, 1993)                                                                         (February 10, 1993)                                  CHO/EP.sub.2                                                                              IFO 50396    FERM BP-4182                                         (Example 4) (January 28, 1993)                                                                         (February 10, 1993)                                  ______________________________________                                    

EXAMPLE 1

(1) Amplification of mouse cDNA fragment having base sequence homologyto human TXA₂ receptor cDNA by the PCR (polymerase chain reaction)method

A single-stranded cDNA was synthesized from mouse lung total RNA byusing random hexanucleotides as primers. PCR primers were designed basedon the human TXA₂ receptor cDNA (HPL) sequences corresponding to theputative third and sixth transmenbrane domains of the receptor [M.Hirata et al., Nature 349, 617 (1991)] Mouse lung cDNA served astemplate in 30 cycles of PCR with 1 min of denaturation at 95° C., 0.5min of annealing at 60° C., and 1.5 min of extension at 72° C. on aZymoreactor (Atto Corp., Tokyo, Japan). A single 418-base pair cDNAfragment was amplified and subcloned into pBluescript SK(+)(Stratagene). A clone isolated (LT3) showed a sequence 78% homologous tothe corresponding region of the human cDNA (HPL).

(2) Cloning mouse prostaglandin E₂ receptor (EP_(3')) cDNA

Mouse lung cDNA prepared by an oligo (dT) priming method wassize-selected (>1.8 kilobases) and inserted into the EcoRI site of λZAPf[DNA (Stratagene) with EcoRI adaptors (New England Biolabs, Inc.).The 1.9×10⁵ clones derived from the cDNA library were screened byhybridization with LT3 obtained in (1) above. Hybridization was carriedout at 58° C. in 6×SSC (900 mM NaCl and 90 mM sodium citrate) containing5×Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 0.1%bovine serum albumin) and 0.5% sodium dodecyl sulfate,and filters werewashed at 60° C. in 2×SSC containing 1% sodium dodecyl sulfate. Amongseveral clones hybridizing positively to LT3, we picked up one (ML64)showing a signal apparently weaker than others. Using this clone as ahybridization probe, the eDNA library of mouse mastocytoma P-815 cellswas screened for a full-length clone. From 7.2×105 clones of the P-815 λZAPII library, nine clones were isolated and subjected to sequenceanalysis. Nucleotide sequencing was carried out on double-strandedtemplates using the dideoxy chain termination method. A full-length DNAclone having a 1,095 bp open reading frame, MP660, was thus obtained.FIG. 1 shows the base sequence (SEQ ID NO:1) of the eDNA of MP660 andthe amino acid sequence (SEQ ID NO:2) deduced therefrom. With respect tothe amino acid sequence, the overlined portions, the sites marked with ★and the sites marked with  denote transmembrane domains I through VII,extracellular domain N-glycosylation sites and sites of phosphorylationby cAMP-dependent protein kinase, respectively.

(3) eDNA expression in COS-1 cells and ligand-binding test

The cDNA of MP660 was cleaved out with EcoRI and inserted to pcDNAI(Invitrogen) and subcloned, followed by transfection of this plasmid DNAto COS-1 cells by the DEAE-dextran method [D. J. Sussmann and G. Milman,Mol. Cell. Biol., 4, 1641 (1984)]. After 72-hour cultivation, cells wereharvested and cell membranes were separated [M. Hirata et al., Nature,349, 617 (1991)]. Using these cell membranes, various [3H]-labeledprostaglandins were assayed for binding activity; [3H]-PGE₂ was found toshow specific binding. Also, the PGE₂ receptor obtained was identifiedas the subtype EP₃α receptor. FIG. 2 shows inhibitory activities ofvarious ligands on the binding of [³ H]-PGE₂ to the MP660-transfectedcell membrane [panel "a" is for inhibitory activities of variousprostaglandins (◯: PGE₂ ; : PGE₁ ; ▪: iloprost; Δ: PGF₂α ; □: PGD₂);panel "b" is for inhibitory activities of prostaglandin-like substances(▴: M&B28,767; : GR63799X; Δ: butaprost; ◯: SC-19220)]. Specificity ofthis binding is shown in FIG. 2a. The binding of [³ H]PGE₂ was inhibitedby unlabeled PGs in the order of PGE₂ =PGE₁ >iloprost, a PGI₂analogue>PGF₂α >PGD₂. Because PGE receptor is pharmacologicallysubdivided into three receptor subtypes, EP₁, EP₂, and EP₃, withdifferent agonist and antagonist profiles, the specificity of this [³H]PGE₂ binding using ligands specific for PGE receptor subtypes wasfurther characterized. As shown in FIG. 2b, among various PGE analogues,only EP₃ -specific agonists, GR 63799X and M&B 28,767, specificallycompeted for the[³ H]PGE₂ binding with equal potency, and they were morepotent than PGE₂ itself. On the other hand, no competition was found atall with either an EP₁ -specific antagonist, SC-19220, or an EP₂-specific agonist, butaprost. [³ H]PGE₂ did not bind to membranes ofuntransfected cells. These results established that MP660 encodes theEPs subtype of PGE receptor.

(4) Stable expression and cAMP assay of receptor gene

To obtain cells that stably express the receptor gene, cDNA transfectionwas conducted by the method described by Nakajima et al. [J. Biol.Chem., 267, 2437 (1992)] to establish a cell line. Specifically, theEcoRI fragment of MP660 was inserted to pdKCR-dhfr [S. Oikawa et al.,Biochem. Biophys. Res. Commun., 164, 39 (1989)], a eukaryotic cellexpression vector having the mouse dhfr gene as a selection marker. Thisplasmid was transfected to CHO-dhfr-(lacking dihydrofolate reductaseactivity) cells [G. Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci. USA,77, 4216 (1980)] by the calcium phosphate method [F. L. Graham and A. J.van der Eb, Virology, 52, 456 (1973)]. The cells were subjected toselection culture in α-modified Eagle's medium [S. Oikawa et al.,Biochem. Biophys. Res. Commun., 164, 39 (1989)] which contained noribonucleoside and deoxyribonucleoside and which contained. 100 unit/mlpenicillin, 100 μg/ml streptomycin and 10% dialyzed bovine fetal serum(Cell Culture Laboratories). The cells which proliferated were clonedand clone cells were obtained. EP₃α receptor cDNA transfection wasconfirmed by the RNA blotting method. Using thus obtained CHO cellswhich constantly express the EP₃α receptor [MP660/KCR cells; IF050366;FERM BP-3803], the effect of PGE₂ on forskolin-stimulated cAIV[Psynthesis or M&B28,767, an EP₃ -specific agonist was assessed. It wasthus found that cAMP synthesis in MP660/KCR cells is inhibited by thecopresence of these substances (see FIG. 3; : PGE₂ ; □: M&B28,767). Asshown in FIG. 3, the transfected CHO cells showed a dosedependentdecrease to PGE₂ in forskolin-induced cellular cAMP accumulation. M&B28,767, an EP₃ -specific agonist, also inhibited forskolin-induced cAMPsynthesis and was more potent than PGE₂ (IC50 of M&B 28,767=1×10⁻¹² M;IC₅₀ of PGE₂ =1×10⁻¹⁰ M).

(5) mRNA expression in various tissues

Total RNAs from various mouse tissues were isolated by the acidguanidinium thiocyanate-phenol-chloroform method [P. Chomczynski and N.Sacchi, Anal. Biochem., 162, 156 (1987)]. Next, from this total RNAs,poly(A)+ RNAs were purified using Oligotex dT₃₀ (Takara Shuzo, Kyoto,Japan). Poly (A)⁺ RNAs (10 μg) from each tissue were separated byelectrophoresis on a 1.2% agarose gel, transferred onto nylon membranes(Hybond-N, Amersham Corp.), and hybridized with a ³² P-labeled EcoRI/BamHI fragment of MP660 clone. Hybridization was carried out at 68° C. in6×SSC, and filters were washed at 68° C. in 1×SSC. Eventually, a 2.3 kbstrong band appeared from tissues on which PGE₂ is pharmacologicallyactive, such as the kidney, stomach and uterus, and from P-815 cells.Another band appeared near 7.0 kb from these tissues and cells.

EXAMPLE 2

(1) Cloning mouse prostaglandin E₂ receptor (EP₃β) cDNA

In substantially the same screening method as in Example 1, using ML64as a hybridization probe, several clones were isolated from mousemastocytoma P-815 cDNA library. Restriction analysis of the isolatedclones displayed at least two types of cDNAs, one type represented byMP660 obtained in Example 1 and another type represented by MP653.Sequencing analyses revealed that MP653 had a 1,083 base pair (bp) openreading frame. FIG. 4 shows the base sequence (SEQ ID NO:3) of the cDNAof MP653 and the amino acid sequence (SEQ ID NO:4) deduced therefrom ascompared with those of MP660. MP653 is identical to MP660 in thenucleotide sequence except deletion of an 89-bp sequence in the codingregion of the putative C-terminal tail of the receptor in MP660-encodedreceptor (FIG. 5). Deletion of this 89-bp sequence creates anotherreading frame downstream from this junction, which extends coding regionuntil a new stop codon placed on 77-bp downstream from the stop codon ofMP660. As a consequence, a 30-amino-acid (aa) C-terminal fragment of theMP660-encoded receptor (peptide-α) was replaced with a new 26-anfragment (peptide-β) in the C-terminal end of MP653-encoded receptor.

(2) Expression of the MP653 cDNA in COS-1 cells and ligand-binding assay

The cDNA of MP653 was cleaved out with EcoRI and inserted to pcDNAI(Invitrogen) and subcloned, followed by transfection of this plasmid DNAto COS-1 cells by the DEAE-dextran method [D. J. Sussmann and G. Milman,Mol. Cell. Biol., 4, 1641 (1984)]. After 72-hour cultivation, cells wereharvested and cell membranes were separated [M. Hirata et al., Nature,349, 617 (1991)]. Using these cell membranes, various [³ H]-labeledprostaglandins were assayed for binding activity; [³ H]-PGE₂ was foundto show specific binding. Also, the PGE₂ receptor obtained wasidentified as the subtype EP₃β receptor. The result of inhibitoryactivities of various ligands on the binding of [³ H]-PGE₂ to theMP653-transfected cell membrance was substantially the same as on thebinding of [³ H]-PGE₂ to the MP660-transfected cell membrance obtainedin Example 1.

MP660-encoding receptor is designated as EP₃α (containing the peptide-α)and MP653-encoding one as EP₃β (containing the peptide-β).

(3) Stable expression and cAMP assay of receptor gene

To obtain cells that stably express the receptor gene, cDNA transfectionwas conducted by the method described by Nakajima et al. [J. Biol.Chem., 267, 2437 (1992)] to establish a cell line. Specifically, theEcoRI fragment of MP653 was inserted to pdKCR-dhfr [S. Oikawa et al.,Biochem. Biophys. Res. Commun., 164, 39 (1989)], a eukaryotic cellexpression vector having the mouse dhfr gene as a selection marker. Thisplasmid was transfected to CHO-dhfr-(lacking dihydrofolate reductaseactivity) cells [G. Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci. USA,77, 4216 (1980)] by the calcium phosphate method IF. L. Graham and A. J.van der Eb, Virology, 52, 456 (1973)]. The cells were subjected toselection culture in α-modified Eagle's medium IS. Oikawa et al.,Biochem. Biophys. Res. Commun., 164, 39 (1989)] which contained noribonucleoside and deoxyribonucleoside and which contained 100 unit/mlpenicillin, 100 μg/ml streptomycin and 10% dialyzed bovine fetal serum(Cell Culture Laboratories). The cells which proliferated were cloned tohave clone cells. EP₃β receptor cDNA transfection was confirmed by theRNA blotting method. Using thus-obtained CHO cells which constantlyexpress the EP₃β receptor [MP653/KCR cells; IFO 50397, FERM BP-4183],the effect of PGE₂ on forskolin-stimulated cAMP synthesis or M&B28, 767,an EP₃ -specific agonist was assessed. It was thus found that cAMPsynthesis in MP653/KCR cells is inhibited by the co-presence of thesesubstances.

EXAMPLE 3

Expression of EP₃α and EP₃β in various tissues

Measurement of the relative abundance of the two isoforms, EP₃α andEP₃β, expressed in each tissue was performed according to the method ofWang et al. (Proc. Natl. Acad. Sci. 86, 9717 (1989)). Total RNA wasisolated according to Example 1 (5) and the RNAs were transcribed intocDNA by random hexanucleotide priming method using Moloney murineleukemia virus reverse transcriptase (Bethesda Research Laboratories).Each cDNA derived from 2.5 μg RNA was used as template in a PCR withprimers corresponding to nucleotide positions 651-680 (PCR I) and1264-1293 (PCR II). The 5'-end ³² P-labeled PCR II (0.3 pmol; 1.0×10⁶c.p.m./pmol) was incubated in each PCR reaction (final 25 μl).Twenty-three cycles of PCR were performed using the followingtemperature profile: 94° C., 40 s; 60° C., 40 s; 72° C., 1.5 min.DNA-resolved gel was dried and subject to autoradiography, and theradioactivity of the gel corresponding to the bands was counted.Consequently, it was found that in any tissue expressing EP₃, EP₃α wasdominantly expressed over EP₃β.

EXAMPLE 4

(1) Cloning mouse prostaglandin E₂ receptor (EP₂) cDNA

Mouse mastocytoma P-815 cell cDNA library carring cDNAs larger than 2.0kb was prepared according to Example 1 (2). The probe DNA was preparedby PCR using mouse EPs cDNA as a template; this 482 bp fragment coversthe transmembrane segments I-IV region of the EP₃ receptor. The 2.0 ×10⁵clones derived from the cDNA library were screened under either high(Sambrook) or low stringency condition. The resultant positive cloneswere subjected to PCR, restriction and sequence analyses and classifiedinto two major groups; one group (six clones) belonged to EP₃ receptoreDNA, and the other (five clones) showed a sequence homologous but notidentical to EP₃ cDNA. One representative clone (MP412) of the lattergroup, which contains a 1539-base pair open reading frame. FIG. 6 showsthe base sequence (SEQ ID NO:5) of the cDNA of MP412 and the amino acidsequence (SEQ ID NO:6) deduced therefrom. This cDNA was transfected intoCOS-1 cells according to the same manner as in Example 1(3).

(2) PGE₂ Binding and cAMP Assays in COS-1 Cells Expressing MP412 cDNA

After the COS-1 cells carting the plasmid DNA obtained in above (1) werecultured for 72 h, cells were harvested and cell membranes wereprepared. Using these cell membranes, various [³ H]-labeledprostaglandins were assayed for binding activity; [³ H]-PGE₂ was foundto show specific binding. Specific [³ H]-PGE₂ binding to the membrane ofuntransfected cells was almost negligible. FIG. 7 shows the specificityof this binding. Specific [³ H]-PGE₂ binding was inhibited by unlabeledPG in the order of PGE₂ (◯)=PGE₁ ()>>iloprost(▪), a stable PGI2analogue≧PGF₂α (Δ)≧PGD₂ (□). FIG. 8 shows ligand binding specificityusing several ligands which show characteristic agonist or antagonistactivity for PGE receptor subtypes. As shown in FIG. 8, the PGE₂ bindingwas inhibited by misoprostol(), an EP₂ and EP₃ agonist, and more weaklyby M&B 28,767(▴), an EP₃ agonist. On the other hand, sulprostone (□), anEP₁ and EP₃ agonist, SC-19220(◯), an EP₁ antagonist, and butaprost (A),an EP₂ agonist, did not inhibit it. The ability of misoprostol toinhibit PGE₂ binding and no ability of sulprostone suggest that MP412encodes the EP₂ subtype of PGE receptor, and this was also supported byweak cross-reaction of M&B 28,767 to EP₂ (Lawrence, R. A. et al, Br. J.Pharmacol. 105, 271-278 (1992)). The lack of binding activity ofbutaprost in mouse EP₂ might indicate that the action of butaprost isspecies specific or there may be other forms of EP₂ receptor subtype.

EP₂ receptor is coupled to stimulation of adenylate cyclase. For cAMPassay, the plasmid DNA was transfected into COS-1 cells by thelipofection method (P. L. Felgner et al., Proc. Natl. Acad. Sci., 84,7413 (1987)) and cultured for 72 h in a 24-well plate. Cyclic AMP levelsin the cells were determined according to the method of Nakajima et al.(J. Biol. Chem. 267, 2437 (1992)). As shown in FIG. 9, PGE₂dose-dependently increased cAMP level in these cells (:MP412-transfected COS-1 cells; ◯: untransfected COS-1 cells). On theother hand, PGE₂ neither inhibited forskolin-induced cAMP formation andnor accumulated inositol phosphates. These results demonstrate that thisreceptor is an EP₂ subtype coupled exclusively to stimulation ofadenylate cyclase.

To obtain cells that stably express the receptor gene, cDNA transfectionwas conducted by the method according to Example 1 (4) to establish acell line. Specifically, the EcoRI fragment of MP412 was inserted topdKCR-dhfr, a eukaryotic cell expression vector having the mouse dhfrgene as a selection marker. This plasmid was transfected to CHO-dhfr-(lacking dihydrofolate reductase activity) cells. The cells weresubjected to selection culture, the cells which proliferated were clonedto have clone cells, and thus the CHO cells which constantly express theEP₂ receptor [CHO/EP₂ cells; IFO 50396, FERM BP-4182] were obtained.

(3) mRNA expression in various tissues

Poly (A)⁺ RNAs (10 μg) from each tissue, which Were prepared by the samemethod as in Example 1 (5), were separated by electrophoresis on a 1.2%agarose gel, transferred onto nylon membranes (Hybond-N, AmershamCorp.), and hybridized with a ³² P-labeled EcoRI/BamHI fragment of MP412clone. Hybridization was carried out at 68° C. in 6×SSC, and filterswere washed at 68° C. in 2×SSC. A positive band is observed at 3.9kilobase in most of tissues, suggesting widespread distribution of theEP₂ receptor. The tissues highly expressing EP₂ mRNA were ileum andthymus in which PGE₂ induces relaxation of ileum circular muscle andinhibits proliferation of T cells by increasing intracellular cAMPlevels. A significant band was also observed in lung, spleen, heart oruterus. On the other hand, EP₂ mRNA was not detectable in testis andliver.

Other embodiments of the invention will be apparent in the art fromconsideration of the specification and practice of the inventiondisclosed herein. It is intended that the specification and examples beconsidered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2107 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCGGCGGGCGATGGAGAGCAGAGCCTGGGCTCCGGCTGTCCCCCAGTGCACTCTGCTGCT60                ATCCCGCAGCTGAGCCGGGAGGCTCCGGCCCCGTGCGCCCTACCGTGGCCCCGCCACTAT120               GGCTAGCATGTGGGCGCCGGAGCACTCTGCTGAAGCGCACAGCAACCTGTCAAGTACTAC180               CGACGACTGCGGCTCCGTGTCCGTGGCCTTTCCCATCACCATGATGGTCACTGGCTTCGT240               GGGCAACGCGCTGGCCATGCTGCTCGTGTCGCGCAGCTACCGGCGCCGCGAGAGCAAGCG300               CAAGAAGTCTTTCCTGCTGTGCATTGGCTGGCTGGCGCTCACCGACTTAGTGGGGCAGCT360               CCTGACCAGCCCGGTGGTCATCCTCGTGTACCTGTCACAGCGACGCTGGGAGCAGCTCGA420               CCCATCGGGGCGTCTGTGCACCTTCTTCGGGCTAACCATGACAGTGTTCGGGCTATCCTC480               GCTCCTGGTGGCCAGCGCCATGGCCGTGGAGCGCGCCCTGGCCATCCGTGCGCCGCACTG540               GTATGCCAGCCACATGAAGACTCGCGCCACGCCGGTACTGCTGGGCGTGTGGCTGTCTGT600               GCTCGCCTTCGCGCTGCTGCCGGTGCTGGGCGTGGGCCGCTACAGCGTGCAGTGGCCGGG660               CACGTGGTGCTTCATCAGCACCGGGCCGGCGGGCAACGAGACAGACCCTGCGCGCGAGCC720               GGGCAGCGTGGCCTTTGCCTCCGCCTTCGCCTGCTTGGGCTTGCTGGCTCTGGTGGTGAC780               CTTTGCCTGCAACCTGGCGACCATCAAAGCCCTGGTGTCCCGCTGTCGGGCCAAAGCCGC840               CGTCTCGCAGTCCAGCGCCCAGTGGGGCAGAATCACCACGGAGACGGCCATCCAGCTCAT900               GGGGATCATGTGTGTGCTGTCCGTCTGTTGGTCGCCGCTATTGATAATGATGTTGAAAAT960               GATCTTCAATCAGATGTCGGTTGAGCAATGCAAGACACAGATGGGAAAGGAGAAGGAGTG1020              CAATTCCTTTCTAATTGCAGTTCGCCTGGCTTCGCTGAACCAGATCTTGGATCCCTGGGT1080              TTATCTGCTGCTAAGAAAGATCCTTCTTCGGAAGTTCTGCCAGATCAGAGACCACACCAA1140              CTATGCTTCCAGCTCCACCTCCTTGCCCTGCCCAGGCTCCTCAGCCCTGATGTGGAGTGA1200              CCAGCTGGAAAGATGATGAACAACCTGAAGTGGACTTTCATTGCAGTACCTGTTTCCCTG1260              GGTCTGAGAATTTCTTCTCCCAGGGAAGGATGACTGAGTATTTTGGATTGTATCTTCTTT1320              TGGCCTCAATTTTAAGTTTTCCTTGCCATTAAACACACCGAGACAAGCTTTCTTAGGATA1380              ATCTGAGAGTCTGGTTGTTAGCTGGTTCCTGTGAAGACTGAAGACTCTGCACTTGAGACG1440              GGGGCAAGACGACACAGAGCAGCATGGAGAGACTCAGTGCAGAAATATCTCCAGCCTCAG1500              AACCTTTGTGGACATGGACACCTTCATGTATTGATAGTCTGACTCTTCTAAATAGGTCTG1560              AAAAAGCAGCATAAGTTTTTAAACAGTGAAGCATCAATGTGTTGAGAGCAAATGTTCATC1620              TAATAAGCCATGAGCCAAACAAGACAAAAAGTCTACATGAGAGGCAAGAGAGATTCTGCA1680              AAGGGTATTTGTGCCAAGAAGGTATACAGTACCACAGAGTTGTGTCCTCAGTGAGAGTGG1740              GAAATAAGTTTCTAATTTAATTCTAATTACTGGCTCCTCAGTAATTCAGGAATCGTGCCA1800              TCATTTCCCTGCTTTTAAAGGGAGAAGTTTAGCTAAAGACACATTCCAGGTGTCACTAAC1860              AGTTCCAAAGCTAGGTGACTAAATGTTCAGCTAGAGCTGTTAAAAGGAAAACCAGCTAAT1920              TATCATTCCAGTCCAATGCTATTTTTGAATTACTATCTACTTAAGATTTCTCATAATTTG1980              TGCTCAGGCAGCACAATAAAAAGGGGGGGGCAAAATTACTAAGTGACAGTTATTCTGCAT2040              CTAAGTCTGTGACTTTTTTATGAAATAAAATGATTTTGTCTGTGTTGAAATAAAAAAAAA2100              AAAAAAA2107                                                                   (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 365 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaSerMetTrpAlaProGluHisSerAlaGluAlaHisSerAsn                              151015                                                                        LeuSerSerThrThrAspAspCysGlySerValSerValAlaPhePro                              202530                                                                        IleThrMetMetValThrGlyPheValGlyAsnAlaLeuAlaMetLeu                              354045                                                                        LeuValSerArgSerTyrArgArgArgGluSerLysArgLysLysSer                              505560                                                                        PheLeuLeuCysIleGlyTrpLeuAlaLeuThrAspLeuValGlyGln                              65707580                                                                      LeuLeuThrSerProValValIleLeuValTyrLeuSerGlnArgArg                              859095                                                                        TrpGluGlnLeuAspProSerGlyArgLeuCysThrPhePheGlyLeu                              100105110                                                                     ThrMetThrValPheGlyLeuSerSerLeuLeuValAlaSerAlaMet                              115120125                                                                     AlaValGluArgAlaLeuAlaIleArgAlaProHisTrpTyrAlaSer                              130135140                                                                     HisMetLysThrArgAlaThrProValLeuLeuGlyValTrpLeuSer                              145150155160                                                                  ValLeuAlaPheAlaLeuLeuProValLeuGlyValGlyArgTyrSer                              165170175                                                                     ValGlnTrpProGlyThrTrpCysPheIleSerThrGlyProAlaGly                              180185190                                                                     AsnGluThrAspProAlaArgGluProGlySerValAlaPheAlaSer                              195200205                                                                     AlaPheAlaCysLeuGlyLeuLeuAlaLeuValValThrPheAlaCys                              210215220                                                                     AsnLeuAlaThrIleLysAlaLeuValSerArgCysArgAlaLysAla                              225230235240                                                                  AlaValSerGlnSerSerAlaGlnTrpGlyArgIleThrThrGluThr                              245250255                                                                     AlaIleGlnLeuMetGlyIleMetCysValLeuSerValCysTrpSer                              260265270                                                                     ProLeuLeuIleMetMetLeuLysMetIlePheAsnGlnMetSerVal                              275280285                                                                     GluGlnCysLysThrGlnMetGlyLysGluLysGluCysAsnSerPhe                              290295300                                                                     LeuIleAlaValArgLeuAlaSerLeuAsnGlnIleLeuAspProTrp                              305310315320                                                                  ValTyrLeuLeuLeuArgLysIleLeuLeuArgLysPheCysGlnIle                              325330335                                                                     ArgAspHisThrAsnTyrAlaSerSerSerThrSerLeuProCysPro                              340345350                                                                     GlySerSerAlaLeuMetTrpSerAspGlnLeuGluArg                                       355360365                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1405 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAGAGCAGAGCCTGGGCTCCGGCTGTCCCCCAGTGCACTCTGCTGCTATCCCGCAGCTGA60                GCCGGGAGGCTCCGGCCCCGTGCGCCCTACCGTGGCCCCGCCACTATGGCTAGCATGTGG120               GCGCCGGAGCACTCTGCTGAAGCGCACAGCAACCTGTCAAGTACTACCGACGACTGCGGC180               TCCGTGTCCGTGGCCTTTCCCATCACCATGATGGTCACTGGCTTCGTGGGCAACGCGCTG240               GCCATGCTGCTCGTGTCGCGCAGCTACCGGCGCCGCGAGAGCAAGCGCAAGAAGTCTTTC300               CTGCTGTGCATTGGCTGGCTGGCGCTCACCGACTTAGTGGGGCAGCTCCTGACCAGCCCG360               GTGGTCATCCTCGTGTACCTGTCACAGCGACGCTGGGAGCAGCTCGACCCATCGGGGCGT420               CTGTGCACCTTCTTCGGGCTAACCATGACAGTGTTCGGGCTATCCTCGCTCCTGGTGGCC480               AGCGCCATGGCCGTGGAGCGCGCCCTGGCCATCCGTGCGCCGCACTGGTATGCCAGCCAC540               ATGAAGACTCGCGCCACGCCGGTACTGCTGGGCGTGTGGCTGTCTGTGCTCGCCTTCGCG600               CTGCTGCCGGTGCTGGGCGTGGGCCGCTACAGCGTGCAGTGGCCGGGCACGTGGTGCTTC660               ATCAGCACCGGGCCGGCGGGCAACGAGACAGACCCTGCGCGCGAGCCGGGCAGCGTGGCC720               TTTGCCTCCGCCTTCGCCTGCTTGGGCTTGCTGGCTCTGGTGGTGACCTTTGCCTGCAAC780               CTGGCGACCATCAAAGCCCTGGTGTCCCGCTGTCGGGCCAAAGCCGCCGTCTCGCAGTCC840               AGCGCCCAGTGGGGCAGAATCACCACGGAGACGGCCATCCAGCTCATGGGGATCATGTGT900               GTGCTGTCCGTCTGTTGGTCGCCGCTATTGATAATGATGTTGAAAATGATCTTCAATCAG960               ATGTCGGTTGAGCAATGCAAGACACAGATGGGAAAGGAGAAGGAGTGCAATTCCTTTCTA1020              ATTGCAGTTCGCCTGGCTTCGCTGAACCAGATCTTGGATCCCTGGGTTTATCTGCTGCTA1080              AGAAAGATCCTTCTTCGGAAGTTCTGCCAGATGATGAACAACCTGAAGTGGACTTTCATT1140              GCAGTACCTGTTTCCCTGGGTCTGAGAATTTCTTCTCCCAGGGAAGGATGACTGAGTATT1200              TTGGATTGTATCTTCTTTTGGCCTCAATTTTAAGTTTTCCTTGCCATTAAACACACCGAG1260              ACAAGCTTTCTTAGGATAATCTGAGAGTCTGGTTGTTAGCTGGTTCCTGTGAAGACTGAA1320              GACTCTGCACTTGAGACGGGGGCAAGACGACACAGAGCAGCATGGAGAGACTCAGTGCAG1380              AAATATCTCCAGCCTCAGAACCTTT1405                                                 (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 361 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaSerMetTrpAlaProGluHisSerAlaGluAlaHisSerAsn                              151015                                                                        LeuSerSerThrThrAspAspCysGlySerValSerValAlaPhePro                              202530                                                                        IleThrMetMetValThrGlyPheValGlyAsnAlaLeuAlaMetLeu                              354045                                                                        LeuValSerArgSerTyrArgArgArgGluSerLysArgLysLysSer                              505560                                                                        PheLeuLeuCysIleGlyTrpLeuAlaLeuThrAspLeuValGlyGln                              65707580                                                                      LeuLeuThrSerProValValIleLeuValTyrLeuSerGlnArgArg                              859095                                                                        TrpGluGlnLeuAspProSerGlyArgLeuCysThrPhePheGlyLeu                              100105110                                                                     ThrMetThrValPheGlyLeuSerSerLeuLeuValAlaSerAlaMet                              115120125                                                                     AlaValGluArgAlaLeuAlaIleArgAlaProHisTrpTyrAlaSer                              130135140                                                                     HisMetLysThrArgAlaThrProValLeuLeuGlyValTrpLeuSer                              145150155160                                                                  ValLeuAlaPheAlaLeuLeuProValLeuGlyValGlyArgTyrSer                              165170175                                                                     ValGlnTrpProGlyThrTrpCysPheIleSerThrGlyProAlaGly                              180185190                                                                     AsnGluThrAspProAlaArgGluProGlySerValAlaPheAlaSer                              195200205                                                                     AlaPheAlaCysLeuGlyLeuLeuAlaLeuValValThrPheAlaCys                              210215220                                                                     AsnLeuAlaThrIleLysAlaLeuValSerArgCysArgAlaLysAla                              225230235240                                                                  AlaValSerGlnSerSerAlaGlnTrpGlyArgIleThrThrGluThr                              245250255                                                                     AlaIleGlnLeuMetGlyIleMetCysValLeuSerValCysTrpSer                              260265270                                                                     ProLeuLeuIleMetMetLeuLysMetIlePheAsnGlnMetSerVal                              275280285                                                                     GluGlnCysLysThrGlnMetGlyLysGluLysGluCysAsnSerPhe                              290295300                                                                     LeuIleAlaValArgLeuAlaSerLeuAsnGlnIleLeuAspProTrp                              305310315320                                                                  ValTyrLeuLeuLeuArgLysIleLeuLeuArgLysPheCysGlnMet                              325330335                                                                     MetAsnAsnLeuLysTrpThrPheIleAlaValProValSerLeuGly                              340345350                                                                     LeuArgIleSerSerProArgGluGly                                                   355360                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2442 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AGCCTCTCTGGCTTTCCAAGCTTTTTTGAAAGCAAGATACTCTGACCTCAGTTCCGGAAA60                GTTGGCAGCCACCGAGCCCCGGTTCCGAGACAGCAAAAGCTTGACAAGTTCCGCACTGCG120               TGGGAAGAGACTGATGGCTGAGGTTGGAGGTACCATTCCTAGATCGAACCGTGAGCTCCA180               ACGCTGTGTGTTACTAACCACCACCATCATGTCCATCCCCGGAGTCAACGCGTCCTTCTC240               CTCCACTCCGGAGAGGCTGAACAGCCCGGTGACCATTCCCGCAGTGATGTTCATCTTCGG300               GGTGGTGGGCAACCTGGTGGCCATCGTAGTATTGTGCAAGTCGCGCAAGGAGCAGAAAGA360               GACGACCTTTTACACTCTAGTATGTGGGCTGGCTGTCACTGACCTTCTGGGCACCTTGTT420               GGTAAGCCCGGTGACCATCGCCACATACATGAAGGGCCAGTGGCCCGGAGACCAGGCACT480               GTGTGACTATAGCACCTTCATCCTACTTTTCTTCGGTCTGTCGGGTCTCAGCATCATCTG540               TGCCATGAGCATCGAGCGCTACCTGGCCATCAACCACGCCTACTTCTACAGCCACTACGT600               GGACAAGCGGCTGGCCGGCCTCACACTCTTCGCCATCTATGCATCTAACGTGCTGTTCTG660               CGCGCTGCCCAACATGGGCCTGGGCAGATCCGAGCGGCAGTACCCGGGCACCTGGTGCTT720               CATCGACTGGACCACCAACGTAACGGCCTACGCCGCCTTCTCTTACATGTACGCCGGCTT780               CAGCTCCTTCCTCATCCTTGCCACCGTGCTCTGCAACGTGCTGGTGTGCGGCGCGCTGCT840               CCGCATGCACCGCCAGTTCATGCGCCGCACCTCGTTGGGCACGGAGCAGCACCATGCGGC900               TGCCGCCGCCGCGGTAGCTTCGGTGGCCTGTCGGGGCCACGCTGGGGCCTCCCCAGCCCT960               GCAGCGCCTCAGCGACTTTCGCCGCCGCAGGAGTTTCCGGCGCATCGCGGGTGCGGAGAT1020              CCAGATGGTCATCTTACTCATCGCCACCTCTCTGGTGGTGCTCATCTGCTCCATTCCGCT1080              CGTGGTGCGAGTGTTCATTAACCAGTTATATCAGCCAAACGTGGTGAAAGACATCAGCAG1140              AAACCCAGATTTGCAGGCCATCAGGATTGCTTCTGTGAACCCCATCCTGGACCCCTGGAT1200              TTACATCCTTCTTCGGAAGACTGTGCTCAGTAAAGCCATAGAGAAGATCAAGTGCCTCTT1260              CTGCCGCATTGGCGGTTCCGGCAGAGACAGCTCGGCCCAGCACTGCTCAGAGAGTCGGAG1320              GACATCTTCCGCCATGTCCGGCCACTCTCGCTCCTTCCTCGCCCGGGAGTTAAAGGAGAT1380              CAGCAGCACGTCCCAGACCCTCCTGTACCTGCCAGACCTGACTGAAAGCAGCCTCGGAGG1440              CAGGAATTTGCTTCCAGGTTCGCATGGCATGGGCCTGACCCAAGCAGACACCACCTCGCT1500              GAGAACTTTGCGAATTTCCGAGACCTCAGACTCCTCCCAGGGCCAGGACTCTGAGAGTGT1560              CCTGTTGGTGGATGAGGTTAGTGGGAGCCACAGAGAGGAGCCTGCCTCTAAAGGAAACTC1620              TCTGCAAGTCACATTCCCCAGTGAAACTCTGAAATTATCTGAAAAATGTATATAGTAGCT1680              AAAGGGGGAATCTTATAAAATCCTGTGCAATAGACATACATAGCTGTACTCAGAAGGGCT1740              GTCTTCATCTGGACTCCCACTAGAGAACAGGCGAGCTCCTGAGGGCTCTCCAAGGCTGCA1800              GACTGAGGTCCTTGAGTGCCCAGGCTTGAAGCACATTGGCTGTCATTCTGATGTGACTCG1860              AGATTGCAGTTGCAACTTGGCAGCTTTTTTCTACTGGACAGGAAGATGGCAGAAGCTACG1920              CTATTGTCATAGCAAAAGAGCTTTCTATTTGGCACATACCAGGGGTCCAGCTACTGGAAG1980              GGCTCTACCCCAAACTCTGAGGACTACCTTACAGCTGACTTAAGTGTCTCACTAAAGCAT2040              GAAATGTGAATTTTTATTGTTGGAAATATAATTTAAGGTATTTATGTTCTTCTCTGTGAG2100              AAGGTTTATTGTTAATACAAGGTATAAAAAACACATGATATGCCCTCTCCTGCCAATATA2160              ACCAGCTAATATTGTCGATGTTATTTTTTTTTTTCCATAAACAAGTTCAGGCCAAAGTGT2220              TGAAAACAGAGTGAAACTAATATCTATAAAATAGATATAAATTTTTAAAATAGTTTAGTA2280              TCATCAAAGAAAAAATAAGTAGTATTTAAGATGTGAAAAATGAACAACCTAAAATATATT2340              TTCCAAGCTATATATAATAATGAAAAATAAAAACATTACATTTATTTATCCAGAAAACTG2400              TGATTTTAGGATTACCTAACATTGCTGGTAAATATTTTCAAC2442                                (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 513 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetAlaGluValGlyGlyThrIleProArgSerAsnArgGluLeuGln                              151015                                                                        ArgCysValLeuLeuThrThrThrIleMetSerIleProGlyValAsn                              202530                                                                        AlaSerPheSerSerThrProGluArgLeuAsnSerProValThrIle                              354045                                                                        ProAlaValMetPheIlePheGlyValValGlyAsnLeuValAlaIle                              505560                                                                        ValValLeuCysLysSerArgLysGluGlnLysGluThrThrPheTyr                              65707580                                                                      ThrLeuValCysGlyLeuAlaValThrAspLeuLeuGlyThrLeuLeu                              859095                                                                        ValSerProValThrIleAlaThrTyrMetLysGlyGlnTrpProGly                              100105110                                                                     AspGlnAlaLeuCysAspTyrSerThrPheIleLeuLeuPhePheGly                              115120125                                                                     LeuSerGlyLeuSerIleIleCysAlaMetSerIleGluArgTyrLeu                              130135140                                                                     AlaIleAsnHisAlaTyrPheTyrSerHisTyrValAspLysArgLeu                              145150155160                                                                  AlaGlyLeuThrLeuPheAlaIleTyrAlaSerAsnValLeuPheCys                              165170175                                                                     AlaLeuProAsnMetGlyLeuGlyArgSerGluArgGlnTyrProGly                              180185190                                                                     ThrTrpCysPheIleAspTrpThrThrAsnValThrAlaTyrAlaAla                              195200205                                                                     PheSerTyrMetTyrAlaGlyPheSerSerPheLeuIleLeuAlaThr                              210215220                                                                     ValLeuCysAsnValLeuValCysGlyAlaLeuLeuArgMetHisArg                              225230235240                                                                  GlnPheMetArgArgThrSerLeuGlyThrGluGlnHisHisAlaAla                              245250255                                                                     AlaAlaAlaAlaValAlaSerValAlaCysArgGlyHisAlaGlyAla                              260265270                                                                     SerProAlaLeuGlnArgLeuSerAspPheArgArgArgArgSerPhe                              275280285                                                                     ArgArgIleAlaGlyAlaGluIleGlnMetValIleLeuLeuIleAla                              290295300                                                                     ThrSerLeuValValLeuIleCysSerIleProLeuValValArgVal                              305310315320                                                                  PheIleAsnGlnLeuTyrGlnProAsnValValLysAspIleSerArg                              325330335                                                                     AsnProAspLeuGlnAlaIleArgIleAlaSerValAsnProIleLeu                              340345350                                                                     AspProTrpIleTyrIleLeuLeuArgLysThrValLeuSerLysAla                              355360365                                                                     IleGluLysIleLysCysLeuPheCysArgIleGlyGlySerGlyArg                              370375380                                                                     AspSerSerAlaGlnHisCysSerGluSerArgArgThrSerSerAla                              385390395400                                                                  MetSerGlyHisSerArgSerPheLeuAlaArgGluLeuLysGluIle                              405410415                                                                     SerSerThrSerGlnThrLeuLeuTyrLeuProAspLeuThrGluSer                              420425430                                                                     SerLeuGlyGlyArgAsnLeuLeuProGlySerHisGlyMetGlyLeu                              435440445                                                                     ThrGlnAlaAspThrThrSerLeuArgThrLeuArgIleSerGluThr                              450455460                                                                     SerAspSerSerGlnGlyGlnAspSerGluSerValLeuLeuValAsp                              465470475480                                                                  GluValSerGlySerHisArgGluGluProAlaSerLysGlyAsnSer                              485490495                                                                     LeuGlnValThrPheProSerGluThrLeuLysLeuSerGluLysCys                              500505510                                                                     Ile                                                                           __________________________________________________________________________

What is claimed is:
 1. A recombinant DNA coding for a prostaglandin Ereceptor comprising the amino acid sequence set forth in SEQ ID NO:2orSEQ ID NO:6.
 2. A vector comprising the DNA according to claim
 1. 3. Ahost transformed with the vector of claim
 2. 4. A method for producing aprostaglandin E receptor which comprises cultivating the transformedhost of claim 3 in a culture medium under conditions that would allowexpression of the receptor, and recovering the receptor.
 5. The DNAaccording to claim 1, which comprises nucleotides 119 to 1213 of SEQ IDNO:1 or nucleotides 134 to 1672 of SEQ ID NO:5.
 6. The DNA according toclaim 1, which is isolated from mouse tissue.
 7. A recombinant DNAcoding for a prostaglandin E receptor comprising amino acids 1 to 513 ofSEQ ID NO:6.
 8. A recombinant DNA coding for a prostaglandin E receptorcomprising amino acids 1 to 365 of SEQ ID NO:2.